Our long term aim is to use a genetic approach in Drosophila to understand molecular mechanisms of motorneuron fate specification and differentiation. In this proposal, we focus on mechanisms linking motorneuron fate to axon guidance. In a large-scale screen for mutants affecting motorneuron development, we have identified foxO and Mmp2, both of which are expressed in motorneuron subsets and necessary for proper motor axon guidance. We focus on these two evolutionarily-conserved proteins as they provide us with novel entry points for elucidating how distinct behaviors of motorneurons are developmentally regulated. Our goals here are to complete molecular, genetic, and phenotypic analyses of these genes in order to understand the observed mutant phenotypes. FoxO is a transcription factor best known for its role in the insulin signaling pathway where it is regulated by extracellular signals. Our preliminary data suggest that FoxO is expressed specifically in clusters of motorneurons in response to a target-derived signal. Detailed expression and phenotypic analyses will elucidate the role of FoxO in motorneuron development. Furthermore, we will identify the signaling pathway(s) regulating FoxO expression in motorneurons through molecular and genetic epistasis experiments. Matrix metalloproteinases (Mmps) comprise a large family of. transmembrane and secreted proteases that together cleave nearly every component of the ECM.Mmp2 is expressed in stereotyped populations of motorneurons and is necessary for proper motor axon guidance. We will characterize the expression pattern of Mmp2 in post-mitotic neurons and elucidate its role in motor axon guidance by analyzing motor axon outgrowth in Mmp2 mutant embryos. Additionally, we will establish whether Mmp2 is necessary for motor axon defasciculation by analyzing genetic interactions between Mmp2 and guidance molecules known to regulate this key pathfinding step. These studies will advance our understanding of how molecules acting in distinct motorneuron populations coordinate to establish proper patterns of neuromuscular connectivity. Motorneuron differentiation is an essential event in neuronal development and can be disrupted in human development and disease. Furthermore, since both foxO and Mmp2 are evolutionarily-conserved proteins acting in pathways of intense clinical interest, these studies should have broad biological and medical significance.